Transgenesis of the gonochoristic nematode Caenorhabditis inopinata by microparticle bombardment with hygromycin B selection

The gonochoristic nematode Caenorhabditis inopinata is the phylogenetically closest species to the well-studied nematode Caenorhabditis elegans (Kanzaki et al. , 2018). While C. inopinata has been expected to be a useful comparative model for C. elegans , efficient transgenesis methods have not been available. Here, we established a method to integrate transgenes into the C. inopinata genome by microparticle bombardment with hygromycin B selection. C. elegans- derived genetic elements tested in this study, including universal and germline-specific promoters, ORFs, and 3’UTRs, were all functional in C. inopinata. Using this method, transgenic C. inopinata strains that express fluorescent subcellular markers were established.


Description
C. inopinata is a recently isolated sister species to C. elegans (Kanzaki et al., 2018). Despite their phylogenetic proximity, these two species have various morphological and ecological differences in, for example, body length (C. elegans: 1 mm, C. inopinata: 2-3 mm), sex (hermaphroditic vs. gonochoristic), and optimum temperature for growth (20 °C vs. 27 °C). Various microinjection-based transgenesis methods have been developed for C. elegans, including the formation of extrachromosomal arrays (Mello et al., 1991), transposon-or CRISPR/Cas9-based gene integration (Frøkjaer-Jensen et al., 2008;Dickinson et al., 2013). In these methods, transgene-containing plasmids are injected into adult hermaphrodite gonads. In contrast, microinjection in C. inopinata is technically difficult because of the thin and fragile structure of female gonads and the small brood size (average < 20), and transgenesis has been largely unsuccessful. To circumvent this problem, we established a method to integrate transgenes into the C. inopinata genome by microparticle bombardment with hygromycin B selection.
The plasmids for the bombardment were designed to contain a transgene and the hygromycin resistance gene cassette (Fig.  1A). Because of the high level of sequence conservation between C. inopinata and C. elegans, genetic elements derived from C. elegans were applied to express transgenes in C. inopinata (Kanzaki et al., 2018). As promoters, the eft-3 promoter and the rps-0 promoter (both for universal expression; Hunt-Newbury et al., 2007;Frøkjaer-Jensen et al., 2012) and the pie-1 promoter (for germline-specific expression; Praitis et al., 2001) were used. As universal 3'UTRs, elements from tbb-2 or unc-54 were used (Merritt et al., 2008). As for ORFs, C. elegans his-58 (histone H2B), tbb-2 (β-tubulin), a mammalian PH (Pleckstrinhomology) domain (Audhya et al., 2005), and the GFP sequence modified for C. elegans were used.
The protocol for microparticle bombardment and hygromycin B screening for C. inopinata was based on the protocols for C. elegans (Greiss and Chin, 2011;Radman et al., 2013) and P. pacificus (Namai and Sugimoto, 2018) with some modifications ( Fig. 1B and Methods). First, the culture conditions were optimized for C. inopinata. Worms were grown at 27 °C on the NGM plates containing a higher concentration (2.5%) of agar to avoid burrowing, which were seeded with Escherichia coli HT115(DE3). Second, because C. inopinata is gonochoristic, a mating process for propagation was added during the hygromycin B screening. If there were <15 survivors per plate on Day 3 after hygromycin B addition, wild-type adults (mixtures of males and females) were added for mating with the survivors. After successful propagation, hygromycin B screening was repeated. If the number of survivors on Day 3 was large enough to mate among them, the plates were left for several weeks to obtain integrants, as in the case of hermaphroditic nematodes C. elegans and P. pacificus. At least one integrant line was obtained from five to eight shots targeting 2,000 ~ 20,000 worms per shot.
These results demonstrated that microparticle bombardment with hygromycin B screening is useful for transgenesis in the gonochoristic nematode C. inopinata. Since C. elegans-derived genetic elements seem generally functional in C. inopinata, a large collection of transgene constructs of cell-specific and organelle-specific markers developed for C. elegans can be directly utilized to construct C. inopinata marker strains. This transgenesis method and marker strains will enable more advanced genetic comparative analysis between C. elegans and C. inopinata.

Protocol for microparticle bombardment and hybromycin B screening
This microparticle bombardment protocol corresponding to the 5 shots for a single plasmid, is based on the ones developed for other nematode species (Praitis et al., 2001;Greiss and Chin, 2011;Radman et al., 2013;Namai and Sugimoto, 2018) and modified for C. inopinata.

I. Collecting the adult worms for the bombardment
1. Place the 10 mated C. inopinata adult females on 100 NGM 90 mm plates seeded with HT115 and incubate at 27 °C for ~7 days to obtain F2 young adults.
3. Stand the tubes for 10 minutes at room temperature and let most adult worms settle down.
4. Remove the supernatant containing bacteria, eggs, and larvae using a sterile Pasteur pipette. 5. Collect the precipitated adult worms into a single 50 ml conical tube by decantation.
6. Fill up the tube with M9 buffer. 7. Stand the tube for 10 minutes at room temperature and let most adult worms settle down.
8. Remove the supernatant. 9. Repeat washing steps (6 to 8) at least three times until the buffer appears clear of bacteria, eggs, and small larvae.
10. Transfer the adult worms to a 15 mL conical tube.
11. Centrifuge the tube for 1 minute at 800 × g to precipitate the adult worms and remove the supernatant. At this step, 2-5 ml worm suspension can be obtained. Use 1 ml of the worm suspension (2,000 ~ 20,000 worms) for one shot of bombardment.
12. Keep the worm suspension in the 15 mL conical tube at 22 °C horizontally on the shaker at 100 rpm for aeration until bombardment (The incubation period should not exceed 12 hours).

II. Washing gold microcarriers
1. One day after microparticle bombardment, add 10 mg/ml hygromycin B (Invitrogen) onto the plates to a final concentration of 330 μg/ml and dry the plates.